Driven by their impact on human health, the detection and quantification of metal ions and organic molecules in biological and environmental systems has attracted intense attention recently (Nolan and Lippard, Chem. Rev. 2008, 108, 3443-3480; Que et al., Chem. Rev. 2008, 108, 1517-1549). Although instrumental analysis is the routine method of probing these systems, the cost and complicated operation of the required instruments limit their usefulness in carrying out the on-site and real-time detection that is crucial for these systems. To overcome these limitations, a number of highly sensitive and selective sensors have been developed that are portable and offer rapid quantification (Burdette et al., J. Am. Chem. Soc. 2001, 123, 7831-7841; Chang, et al., Proc. Nat. Acad. Sci. U.S.A. 2004, 101, 1129-1134; Chen, P.; He, C. J. Am. Chem. Soc. 2004, 126, 728-729; Yoon et al., J. Am. Chem. Soc. 2005, 127, 16030-16031; Wu et al., Anal. Chem. 2007, 79, 2933-2939; Zhang et al., J. Am. Chem. Soc. 2007, 129, 15448-15449; Chen and He Curr. Opin. Chem. Biol. 2008, 12, 214-221; Xue et al., J. Am. Chem. Soc. 2008, 130, 3244; Wong et al., J. Am. Chem. Soc. 2009, 131, 7142-7152; Xue et al., J. Am. Chem. Soc. 2009, 131, 11668; Xu et al., Angew. Chem., Int. Ed. 2009, 48, 6849-6852; Wu et al., J. Am. Chem. Soc. 2009, 131, 12325-12332). While these results are promising, a more general platform must be developed so that one strategy can be used to select a sensor for any of a wide range of analytes.
One general sensor platform is based on functional nucleic acids (FNAs). These functional molecules (such as DNA or RNA molecules) have been identified through a combinatorial method known as in vitro selection (Breaker et al., Chem. Biol. 1994, 1, 223-229; Robertson and Joyce, Nature 1990, 344:467) or Systematic Evolution of Ligands by Exponential Enrichment (SELEX) (Ellington et al., Nature 1990, 346, 818-822; Tuerk and Gold, Science 1990, 249, 505) from random nucleic acid libraries containing 1014 or more sequences. The catalytic nucleic acids (also called DNAzymes, RNAzymes, deoxyribozymes, ribozymes, RNA enzymes or DNA enzymes), aptazymes, and aptamers selected through these methods are reported to exhibit catalytic activity and binding affinity, respectively in the presence of a diverse number of targets, which range from metal ions and small organic molecules to macromolecules, proteins, nucleic acids, and even viruses and cells (Cuenoud et al., Nature 1995, 375, 611-614; Carmi et al., Chem. Biol. 1996, 3, 1039-1046; Santoro et al., J. Am. Chem. Soc. 2000, 122, 2433-2439; Li et al., Nucleic Acids Res. 2000, 28, 481-488; Li and Lu, J. Am. Chem. Soc. 2000, 122, 10466-10467; Bruesehoff et al., Comb. Chem. High Throughput Screening 2002, 5, 327-335; Mei et al., J. Am. Chem. Soc. 2003, 125, 412-420; Wang and Silverman, J. Am. Chem. Soc. 2003, 125, 6880-6881; Lee et al., Nucleic Acids Res. 2004, 32, D95-D100; Navani and Li. Curr. Opin. Chem. Biol. 2006, 10, 272-281; Liu et al., Proc. Nat. Acad. Sci. U.S.A. 2007, 104, 2056-2061; Song et al., TrAC, Trends Anal. Chem. 2008, 27, 108-117; Liu et al., Chem. Rev. 2009, 109, 1948-1998; Robertson and Joyce, Nature 1990, 344:467; Breaker and Joyce, Chem. Biol. 1994, 1:223; Tuerk and Gold, Science 1990, 249:505; Ellington et al., Nature 1990, 346:818; Huizenga and Szostak, Biochemistry 1995, 34:656; Nutiu and Li, J. Am. Chem. Soc. 2003, 125:4771; Santoro et al., J. Am. Chem. Soc. 2000, 122, 2433; Li et al., Acc. Chem. Res. 2010, 43:631). Unlike other molecules used for sensor design, functional nucleic acids have predictable secondary structures that can be easily functionalized them with fluorophores, chromophores or electrochemical tags, making it possible to transform the specific interactions between functional nucleic acids and their targets into detectable signals (Navani and Li, Curr. Opin. Chem. Biol. 2006, 10, 272-28; Song et al., TrAC, Trends Anal. Chem. 2008, 27, 108-117; Liu et al., Chem. Rev. 2009, 109, 1948-1998; Rajendran and Ellington, Comb. Chem. High Throughput Screening 2002, 5, 263-270; Lu, Chem. Eur. J. 2002, 8, 4588-4596; Willner and Zayats, Angew. Chem., Int. Ed. 2007, 46, 6408-6418; Willner et al., Chem. Soc. Rev. 2008, 37, 1153-1165; Li and Lu, Functional; Springer: New York, 2009; Xiao et al., Angew. Chem., Int. Ed. 2005, 44, 5456-5459; Xiao et al., J. Am. Chem. Soc. 2005, 127, 17990-17991; Baker et al., J. Am. Chem. Soc. 2006, 128, 3138-3139; Zayats et al., J. Am. Chem. Soc. 2006, 128, 13666-13667; Shlyahovsky et al., J. Am. Chem. Soc. 2007, 129, 3814; Zuo et al., J. Am. Chem. Soc. 2007, 129, 1042-104; He, S. J.; Li, D.; Zhu et al., Chem. Com., Zhang, et al., Small 2008, 4, 1196-1200; Schlosser and Li, Chem. Biol. 2009, 16, 311-322; Swensen et al., J. Am. Chem. Soc. 2009, 131, 4262-4266; Zuo et al., J. Am. Chem. Soc. 2009, 131, 6944; Freeman et al., Angew. Chem., Int. Ed. 2009, 48, 7818-7821; Freeman et al., J. Am. Chem. Soc. 2009, 131, 5028). Therefore, numerous functional DNA sensors, such as fluorescent (Shlyahovsky et al., J. Am. Chem. Soc. 2007, 129, 3814; Freeman et al., Angew. Chem., Int. Ed. 2009, 48, 7818-7821; Freeman et al., J. Am. Chem. Soc. 2009, 131, 5028; Nutiu, R.; Li, Y. Chem. Eur. J. 2004, 10, 1868-1876; Cho et al., Top. Fluoresc. Spectrosc. 2005, 10, 127-155; Cao et al., Curr. Proteomics 2005, 2, 31-40; Liu and Lu, Methods Mol. Biol. 2006, 335, 275-288), colorimetric (Liu and Lu, J. Am. Chem. Soc. 2003, 125, 6642-6643; Pavlov et al., J. Am. Chem. Soc. 2004, 126, 11768-11769; Huang et al., Anal. Chem. 2005, 77, 5735-5741; Liu and Lu, Angew. Chem., Int. Ed. 2006, 45, 90-94; Lee et al., Angew. Chem., Int. Ed. 2007, 46, 4093-4096; Lee et al., J. Am. Chem. Soc. 2008, 130, 14217-14226; Wang, Z et al., Adv. Mater. 2008, 20, 3263-3267; Zhao et al., Small 2008, 4, 810-816), and electrochemical (Willner, I.; Zayats, M. Angew. Chem., Int. Ed. 2007, 46, 6408-6418; Zayats et al., J. Am. Chem. Soc. 2006, 128, 13666-13667; Xiao et al., J. Am. Chem. Soc. 2007, 129, 262-263) sensors based on this platform, have been developed. Among them, fluorescent sensors are particularly interesting because of their high sensitivity, simple instrumentation, and reproducible quantification.
Most fluorescent functional DNA sensors require covalent coupling of a fluorophore or a quencher to either the end, or the internal site or the 3′- or 5′-end of a DNA strand. The interaction between a functional DNA and its target induces the separation of the fluorophore and the quencher, causing an observable increase in fluorescence (Nutiu and Li, Chem. Eur. J. 2004, 10, 1868-1876; Cho et al., Top. Fluoresc. Spectrosc. 2005, 10, 127-155; Cao et al., Curr. Proteomics 2005, 2, 31-40). However, DNA labeling can be complicated, expensive, and intrusive. The label might interfere with a functional DNA as it interacts with its targets (Jiang et al., Anal. Chem. 2004, 76, 5230-5235; Wang et al., Anal. Chem. 2005, 77, 3542-3546). In addition, the label can make it difficult to introduce a labeled DNA into a biological system.
To overcome these limitations, label-free fluorescent sensors based on functional DNA have been developed using intercalating dyes (Joseph et al., Biospectroscopy 1996, 2, 173-183; Li, B.; Wei, H.; Dong, S. Chem. Commun. 2007, 73-75; Wang, Y.; Liu, B. Analyst 2008, 133, 1593-1598), malachite green (Babendure et al., J. Am. Chem. Soc. 2003, 125, 14716-14717; Stojanovic et al., J. Am. Chem. Soc. 2004, 126, 9266-9270), and abasic sites (Xu et al., Chem. Commun. 2009, 6445-6447; Xu et al., Chem. Eur. J. 2009, 15, 10375-10378; Xiang et al., J. Am. Chem. Soc. 2009, 131, 15352-15357).